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As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is
committed to providing our customers around the world with the highest quality products.
In addition to direct selling in more than 20 countries in North America, Europe and
Asia/Pacific, Miltenyi Biotec also provides support for our customers through an
extensive distributor network covering dozens of additional countries.

As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is
committed to providing our customers around the world with the highest quality products.
In addition to direct selling in more than 20 countries in North America, Europe and
Asia/Pacific, Miltenyi Biotec also provides support for our customers through an
extensive distributor network covering dozens of additional countries.

Application protocol

In vitro human regulatory T cell suppression assay

This application protocol describes every step from human Treg and Tresp cell isolation, to their co-cultivation in an in vitro suppression assay using the Treg Suppression Inspector, and flow cytometry analysis.

Isolation of PBMCs

Notes:

When working with anticoagulated peripheral blood or buffy coat, PBMCs should be isolated by density gradient centrifugation, for example, using Ficoll-Paque™ according to the manufacturer’s instructions.

Magnetic labeling of non-CD4+ cells

Note:

All steps described below for the magnetic labeling and depletion of non-CD4+ cells, as well as the labeling and selection of CD25+ cells, are performed using the CD4+CD25+ Regulatory T Cell Isolation Kit, human.

Treg and Tresp cells are simultaneously isolated with the CD4+ CD25+ Regulatory T Cell Isolation Kit, human, from one blood sample. The Treg cells (CD4+ CD25+) are the final positive fraction and the Tresp cells (CD4+ CD25–) are the final negative fraction.

Volumes for magnetic labeling given below are for up to 1×10⁷ total cells. When working with fewer than 1×10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2×10⁷ total cells, use twice the volume of all indicated reagent volumes and total volumes).

Adjust the volume to 500 μL with separation buffer.▲ Note: Resuspend up to 1×10⁸ cells in 500 μL of separation buffer. For higher cell numbers, scale up buffer volume accordingly.

Magnetic depletion of non-CD4+ cells

Notes:

The first step of Treg cell isolation is a depletion of non-CD4+ cells. Here, an LD Column is used, which has a capacity of 1×108 labeled cells and 5×108 total cells.

When using the CD4+CD25+ Regulatory T cell Isolation Kit, human, we do not recommend to process more than 1.3×108 total cells on an LD Column. When exceeding this cell number, it is strongly recommended to split the sample and use additional LD Columns.

Place LD Column(s) in the magnetic field of a MidiMACS™ Separator.

Prepare column by rinsing with 2 mL of separation buffer. Always wait until the column reservoir is empty before proceeding to the next step.

Apply cell suspension onto the column.

Collect unlabeled cells that pass through and wash column twice with 1 mL of separation buffer each. Collect total effluent; this is the unlabeled pre-enriched CD4+ cell fraction which is needed for further Treg and Tresp cell isolation.

Determine cell number.

Magnetic labeling of CD25+ cells

Note:

Volumes for magnetic labeling given below are for an initial starting cell number of up to 1×10⁷ total cells. For higher initial cell numbers, scale up all volumes accordingly.

Magnetic separation of CD25+ cells

Note:

The second step during Treg and Tresp cell isolation is positive selection of CD25+ cells. Here, two consecutive MS Columns are used, with a capacity of 1×10⁷ labeled cells. To not exceed the capacity, it is recommended to determine the frequency of CD25+ cells in your cell suspension by flow cytometry beforehand.

To assess the purity of the isolated Treg and Tresp cells, a flow cytometry analysis must be performed. Please refer to"Surface immunofluorescent staining of Treg and Tresp cells and flow cytometry analysis" for a detailed protocol.

Wash column 3 times with 500 μL of separation buffer each. Collect unlabeled cells that pass through and combine with the effluent from step 3.

Remove column from the separator and place it on a suitable collection tube.

Pipette 1 mL of separation buffer onto the column. Immediately flush out magnetically labeled cells by firmly pushing the plunger into the column.

To increase purity of CD4+CD25+ cells, the eluted fraction can be enriched over a second MS Column (recommended). Repeat the magnetic separation procedure described in steps 1 to 6 using a new MS Column. The isolated Treg and Tresp cells are now ready-to-use for in vitro suppression assay.

General

Notes:

To determine the purity of the isolated Treg and Tresp cells, perform the flow cytometry analysis for the final positive (Treg cells) and negative fraction (Tresp cells). It is also recommended to analyze the starting fraction (collect a sample after "Isolation of PBMCs").

Volumes given below are for up to 1×107 cells. When working with fewer cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes, accordingly (e.g., for 2×107 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry.

Purity analysis of Treg and Tresp cells by flow cytometry

Note:

To assess the purity of the isolated Treg and Tresp cells, cells are analyzed by flow cytometry. The analysis should be performed with the cell sample taken in "Isolation of PBMCs" and the cell samples taken in "Magnetic separation of CD25+ cells".

Exclude dead cells from the analysis by using a live/dead cell exclusion marker (e.g., PI) (data not shown).

Analyze the lymphocytes further for their expression of CD4 (x-axis) and CD25 (y-axis) to assess the frequency of CD4+CD25+ Treg cells and of CD4+CD25- Tresp cells in the starting fraction (A in figure below), as well as in the isolated fractions (B in figure below).

Purity of isolated Treg and Tresp cells. CD4+CD25+ Treg cells and CD4+CD25– Tresp cells were isolated from human PBMCs using the CD4+CD25+ Regulatory T Cell Isolation Kit, human. The cells were fluorescently labeled with CD4-VioBlue and CD25-PE before (A) and after (B) separation.

Purity of isolated Treg and Tresp cells.

CD4+CD25+ Treg cells and CD4+CD25– Tresp cells were isolated from human PBMCs using the CD4+CD25+ Regulatory T Cell Isolation Kit, human. The cells were fluorescently labeled with CD4-VioBlue and CD25-PE before (A) and after (B) separation.

General information

Note:

In this protocol one MACSiBead™ Particle per cell (bead-to-cell ratio 1:1) is used for stimulation.

For in vitro suppression, Treg cells, Tresp cells, and the Treg Suppression Inspector (amount of MACSiBead Particles) are co-cultured in different ratios as depicted in the table below in a 96-well flat-bottom plate. To improve the final flow cytometry resolution, the cell numbers can be scaled up accordingly (i.e., 2×105 Tresp cells per well, upscaling of Treg cells and MACSiBead Particles accordingly).

Number of Treg cells, Tresp cells and Treg Suppression Inspector (MACSiBead Particles) per well of a 96-well flat-bottom plate.

Ratio Tresp:Treg

Tresp

Treg

Treg Suppression Inspector

1:0

5×104

-

5×104

1:1

5×104

5×104

1×105

2:1

5×104

2.5×104

7.5×104

4:1

5×104

1.3×104

6.3×104

8:1

5×104

6×103

5.6×104

0:1

–

5×104

5×104

1:0

5×104

–

–

0:1

–

5×104

–

Total

3×105

2×105

4×105

Total triplicates

9×105

6×105

1.2×106

Fluorescent labeling of Tresp cells

Note:

To monitor the proliferation of Tresp cells during the in vitro suppression assay, the cells have to be stained with a fluorescent dye, which allows tracking of cell division, e.g., by using the CellTrace™ Violet Cell Proliferation Kit. For more information please refer to the manufacturer's instruction. It is also possible to use the CSFE-method or the 3H-thymidine incorporation method with this in vitro suppression assay protocol. Data shown in this application protocol were acquired with the CellTrace Violet Cell Proliferation Kit.

Cell preparation

Determine the total number of Treg cells and Tresp cells. For one assay, as outlined in the table in "General information", 9×10⁵ Tresp cells and 6×10⁵ Tregs are needed (if suppression assay is performed with higher cell numbers, scale up the number of cells accordingly).

Resuspend Treg Suppression Inspector in 120 μL of suppression medium. The reagent is now ready to use.▲ Note: Concentration of prepared Treg Suppression Inspector is now 1×10⁷ MACSiBead Particles per mL.

Pipette the appropriate volumes of Treg Suppression Inspector (amount of MACSiBead Particles) to the Treg/Tresp co-cultured cells into the 96-well flat-bottom plate. Refer to the table below for the respective volumes.

Fill up wells to a total volume of 210 µL with suppression medium (see table below).

Pipetting scheme for one assay with a total volume of 210 µL of cell suspension per well with a concentration of 5×105 cells/mL.

Ratio Tresp:Treg

Tresp (5×105 cells/mL)

Treg (5×10⁵ cells/mL)

Treg Suppression Inspector (1×107 MACSiBead Particles/mL)

Culture medium

1:0

100 µL

–

5 µL

105 µL

1:1

100 µL

100 µL

10 µL

–

2:1

100 µL

50 µL

7.5 µL

53 µL

4:1

100 µL

25 µL

6.5 µL

79 µL

8:1

100 µL

12.5 µL

6 µL

92 µL

0:1

–

100 µL

5 µL

105 µL

1:0

100 µL

–

–

110 µL

0:1

–

100 µL

–

110 µL

Total volume

600 µL

387.5 µL

40 µL

654 µL

Total volume triplicates

1800 µL

1200 µL

120 µL

~ 2 mL

Incubation

Incubate the suppression assay at 37 °C and 5–7% CO₂ for 5 days.

▲ Note: If a proliferation dye (e.g. CellTrace or CFSE) was used, refer to "Flow cytometry analysis" for detailed description of final flow cytometry analysis.

▲ Note: If 3H-thymidine was used: after 4 days add the appropriate volume of 3H-thymidine to each well and incubate at 37 °C and 5–7% CO₂ for 16 hours. Measure 3H-thymidine incorporation, e.g., by using a liquid scintillation counter.

Resuspend cell pellet in a suitable amount of separation buffer for analysis by flow cytometry.

Flow cytometry analysis

Identify lymphocytes according to FSC and SSC.

Exclude dead cells from the analysis by using a live/dead cell exclusion marker (e.g., PI).

Gate CD4 cells according to CD4 (y-axis) and FSC (x-axis).

CD4+CD25+ Treg cells can be distinguished from the CD4+CD25– Tresp cells according to CD25 (y-axis) and CellTrace™ (x-axis) (see A in figure below). Gate the CellTrace-positive cells for further cell proliferation analysis (see A in figure below, red gate).

Apply the gating strategy to all samples. Analyze the frequency of proliferating cells using a histogram plot (fluorescence intensity of the CellTrace on the x-axis; see B in figure below) and by determining CellTrace dilution (see B in figure below, red bar).

Final flow cytometry evaluation

Summarized data of an in vitro suppression assay. CD4+ CD25+ Treg cells and CD4+ CD25– Tresp cells were isolated from human PBMCs by using the CD4+ CD25+ Regulatory T Cell Isolation Kit, human and cocultured with the Treg Suppression Inspector in an in vitro suppression assay for 5 days. The suppressive capacity of Treg cells was determined by analyzing the proliferation of Tresp cells under different co-culture conditions.

Summarized data of an in vitro suppression assay.

CD4+ CD25+ Treg cells and CD4+ CD25– Tresp cells were isolated from human PBMCs by using the CD4+ CD25+ Regulatory T Cell Isolation Kit, human and co-cultured with the Treg Suppression Inspector in an in vitro suppression assay for 5 days. The suppressive capacity of Treg cells was determined by analyzing the proliferation of Tresp cells under different co-culture conditions.